Objective-Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes. Approach and Results-In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor β and the inositolrequiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-β family proteins secretion via transcriptional suppression. TGF-β3 but not TGF-β1 or TGF-β2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation. Conclusions-This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-β pathways, leading to neointimal formation. under stress conditions. [21][22][23] Under ER stress conditions, XBP1 mRNA undergoes unconventional splicing through an ER-resident kinase that possesses ribonuclease activity, the inositol-requiring enzyme 1 α (IRE1α). 24 Our recent studies and reports from other groups showed that physiological stimuli such as vascular endothelial cell growth factor could trigger XBP1 splicing in an ER stress response-independent manner. [25][26][27][28] In endothelial cells (ECs), XBP1 splicing plays diverse roles, including cell proliferation, 26 autophagy response, 29 and apoptosis. 30 In SMCs, bone morphogenetic protein-2 was reported to activate XBP1 splicing, 31 but the exact role of XBP1 in SMCs still remains unclear. In this study, we demonstrated that XBP1 splicing is crucial in PDGF-BB-induced SMC proliferation and contributes to neointima formation after vascular injury. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. Results Vascular Injury Increased XBP1 Expression and SplicingIn response to vascular injury, vascular SMCs will be activated undergoing migration, proliferation, and apoptosis. 32 To test whether XBP1 was involved in SMC activation, femoral artery injury model was introduced into the right side of C57Bl/6 mice. The injured vessels were harvested at day 1, and day 3 post surgery, whereas the left uninjured vessels were collected as control. Double immunofluorescence staining with anti-α SMC actin and antispliced XBP1 (XBP1s) or unspliced XBP1 (XBP1u) antibodies were performed on the cryo-sections. As shown in Figure 1A, low levels of XBP1s and XBP1u were detected in the uninjure...